The Impact of Graft Choice on Outcomes Following Pediatric Medial Patellofemoral Ligament Reconstruction: A Systematic Review

The Impact of Graft Choice on Outcomes Following Pediatric Medial Patellofemoral Ligament Reconstruction: A Systematic Review | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Impact of Graft Choice on Outcomes Following Pediatric Medial Patellofemoral Ligament Reconstruction: A Systematic Review Nugroho Tri Wibowo, Tangkas SMHS Sibarani, Rieva Ermawan, Romaniyanto Romaniyanto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8696555/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Successful anterior cruciate ligament (ACL) reconstruction is highly dependent on effective tendon-to-bone healing. Excessive osteoclast activity at the bone–tendon interface can disrupt bone remodeling, impair graft integration, and increase the risk of graft failure. Modulation of osteoclast activity using pharmacological and biological agents may enhance osteointegration after ACL reconstruction. Objective This study aimed to evaluate the effects of alendronate and platelet-rich plasma (PRP), administered alone and in combination, on osteoclast levels at the bone–tendon junction following ACL reconstruction using a calcaneal tendon graft in a sheep model. Methods A randomized controlled experimental study was conducted on 28 sheep subjected to ACL A randomized controlled experimental study was conducted on 28 skeletally mature sheep undergoing ACL reconstruction with a calcaneal tendon autograft. Animals were randomly assigned to four groups (n = 7 per group): control (K0), alendronate-treated (P1), PRP-treated (P2), and combined alendronate plus PRP-treated (P3). Alendronate was administered subcutaneously at a dose of 60 µg/kg weekly for six weeks, while PRP was applied locally at the tendon–bone interface intraoperatively. Histological evaluation was performed 12 weeks postoperatively using hematoxylin–eosin staining. Osteoclasts were identified and quantified at the tendon–bone junction. Data were analyzed using the Kruskal–Wallis test followed by Dunn’s post hoc test. Results here was a significant difference in osteoclast counts among groups (p < 0.0001). The control group exhibited the highest mean osteoclast count (12.00 ± 5.52), followed by the alendronate group (5.43 ± 1.09), PRP group (3.71 ± 0.61), and the lowest count in the combined treatment group (1.71 ± 0.99). Post hoc analysis demonstrated significant differences between all group comparisons (p < 0.05), with the most pronounced reduction observed in the alendronate plus PRP group. Conclusion Alendronate and PRP significantly reduce osteoclast activity at the tendon–bone interface following ACL reconstruction, with a synergistic effect observed when both therapies are combined. These findings suggest that combined alendronate and PRP therapy may enhance tendon-to-bone healing and improve biological conditions for graft integration after ACL reconstruction. Anterior cruciate ligament reconstruction alendronate platelet-rich plasma osteoclast tendon-to-bone healing sheep model Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bone tissue healing and tendon-to-bone integration are complex processes influenced by multiple biological factors. Limited blood supply to ligaments, such as in anterior cruciate ligament (ACL) injuries, impairs spontaneous healing. If left untreated, such injuries can lead to knee instability and secondary cartilage damage, including meniscal injury. A key regulator of bone remodeling is the osteoclast, a multinucleated cell responsible for bone resorption. Excessive osteoclast activity at the tendon insertion site can disrupt tendon–bone integration, weaken the healing interface, and ultimately compromise the success of ligament reconstruction 1 , 2 , 3 . Modulating osteoclast activity is a promising approach to improve tendon-to-bone healing. Studies have shown that suppressing osteoclast function enhances bone formation at the repair site. Alendronate, a bisphosphonate, binds to hydroxyapatite crystals and induces osteoclast apoptosis, thereby reducing bone resorption and promoting faster bone regeneration 4 . In addition, platelet-rich plasma (PRP), a biologic product rich in growth factors such as PDGF and IGF-1, has been reported to suppress osteoclast activity while stimulating osteoblast-mediated bone formation 2 , 5 Although several studies have investigated the effects of alendronate and PRP individually on osteoclast activity, research on their combined use remains limited. This study aims to evaluate the effects of alendronate and PRP on osteoclast levels in a sheep ACL rupture model, providing new insights into biologically driven strategies to accelerate and optimize ACL injury healing and tendon-to-bone integration. Material and Method Study Design This experimental study employed a post-test only control group design using sheep (Ovis aries) as the animal model. Twenty-eight animals were randomly allocated into four groups (n = 7 per group): Control Group (K0): ACL reconstruction using calcaneal tendon graft, followed by subcutaneous administration of 0.9% NaCl. Treatment Group 1 (P1): ACL reconstruction plus weekly subcutaneous injections of alendronate (60 µg/kg body weight) for six weeks. Treatment Group 2 (P2): ACL reconstruction plus local administration of platelet-rich plasma (PRP). Treatment Group 3 (P3): ACL reconstruction plus combined PRP application and weekly alendronate injections (60 µg/kg) for six weeks. All procedures were conducted in accordance with institutional animal welfare regulations and international research ethics standards. Study Setting and Duration The study was conducted at the Animal Laboratory, Faculty of Veterinary Medicine, Universitas Gadjah Mada (Yogyakarta, Indonesia). Histopathological analysis was performed at the Department of Anatomical Pathology, Faculty of Medicine, Universitas Sebelas Maret (Surakarta, Indonesia). The experiment was conducted from April 2025 until completion. Experimental Animals Healthy, skeletally mature male sheep (Ovis aries), aged 2 years and weighing approximately 20 kg, were included. Animals with degenerative joint disease or systemic illness were excluded. Any animal that died during the study period was recorded and excluded from analysis. Sample Size Calculation The minimum sample size was calculated using the Notoadmodjo formula (2018): (𝑡−1) (𝑛−1) > 15 where t = number of groups and n = sample size per group. With four groups, a minimum of six animals per group was required. To account for a potential 10% dropout, the sample size per group was increased to seven, yielding a total of 28 animals. Preoperative Preparation All sheep underwent a 7-day acclimatization period under standard housing conditions and were assessed for general health status. Animals were then randomly assigned to one of the four groups. Surgical Procedure Anterior cruciate ligament (ACL) reconstruction was performed under general anesthesia and sterile conditions. Following a medial parapatellar approach, the native ACL was transected. Autologous calcaneal tendon was harvested, and femoral and tibial tunnels were created anatomically to match the graft diameter. The graft was positioned at the ACL footprint, tensioned at 30° knee flexion, and fixed with a 6-mm biodegradable interference screw. PRP Preparation and Application Autologous whole blood was collected and anticoagulated with sodium citrate. PRP was prepared using a two-step centrifugation protocol to achieve a platelet concentration of at least four times baseline. The prepared PRP was applied intraoperatively around the tendon-bone interface after graft fixation. Alendronate Administration Alendronate was administered subcutaneously at a dose of 60 µg/kg once weekly for six consecutive weeks, starting on the day of surgery. Postoperative Care and Follow-Up Animals were housed under identical environmental and feeding conditions. They were observed daily for wound healing, mobility, and signs of infection or distress. Tissue Harvesting and Histological Analysis At 12 weeks post-surgery, animals were euthanized humanely. The femur–tibia complex was harvested, and tissue samples from the bone–tendon junction were fixed in 10% formalin, decalcified, embedded in paraffin, sectioned, and stained with hematoxylin–eosin (H&E). Osteoclasts were identified as multinucleated cells along the bone surface and counted in five randomly selected high-power fields (HPFs) per section by two independent, blinded pathologists. Outcome Measures The primary outcome measure was the number of osteoclasts at the tendon–bone interface. Statistical Analysis Data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated with Levene’s test. If data were normally distributed and homogeneous, one-way ANOVA was applied followed by Tukey’s post hoc test. Non-normally distributed data were analyzed using the Kruskal–Wallis test followed by Dunn’s multiple comparison test. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using RStudio (version 2024.12.1). Ethical Considerations This study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Sebelas Maret (Approval No. [insert number]). All procedures adhered to the ARRIVE guidelines and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Results Histological examination was successfully performed on all 28 specimens following a 12-week observation period after anterior cruciate ligament reconstruction. Osteoclast evaluation was conducted on sections obtained from the bone–tendon junction and stained using hematoxylin and eosin (H&E). Quantitative assessment of osteoclasts was then performed and the results were compared among the four experimental groups. The mean (± standard deviation) osteoclast counts were highest in the control group (K0: 12.00 ± 5.52). A marked reduction in osteoclast numbers was observed in all intervention groups, with sequentially lower values in the alendronate group (P1: 5.43 ± 1.09), the PRP group (P2: 3.71 ± 0.61), and the lowest count in the combined alendronate and PRP group (P3: 1.71 ± 0.99). These histological differences across groups are illustrated in Figs. 1– 4 . Assessment of data distribution using the Shapiro–Wilk test revealed that osteoclast count data in all groups were not normally distributed (p < 0.05). In addition, Levene’s test demonstrated heterogeneity of variances among groups (p < 0.05). Therefore, the data did not meet the assumptions required for parametric analysis, and nonparametric statistical methods were applied. Group comparisons were performed using the Kruskal–Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. Table 1 Mean of osteoclast number after intervention Group Mean ± Standard Deviation Normality Homogeneity K0 12.00 ± 5.52 0.002 0,0134 P1 5.43 ± 1.09 0.001 P2 3.71 ± 0.61 0.005 P3 1.71 ± 0.99 0.032 The Kruskal–Wallis analysis showed a statistically significant difference in osteoclast counts among the four groups (p < 0.0001), as presented in Table 1 . Further post hoc analysis using Dunn’s test demonstrated significant differences between all pairwise group comparisons (p < 0.05), as summarized in Table 2. These findings indicate that both single-agent interventions (alendronate or PRP) and the combined therapy significantly reduced osteoclast numbers at the tendon–bone interface compared with the control group. Overall, the histological results suggest that modulation of bone remodeling through alendronate and PRP effectively suppresses osteoclastic activity following ACL reconstruction. The most pronounced reduction in osteoclast counts was consistently observed in the combined treatment group (P3), supporting a potential synergistic effect of alendronate and PRP in promoting a more favorable biological environment for tendon-to-bone healing. . Table 2 Statistical analysis results of post hoc Dunn’s test on number of osteoclast Group p-value K0 vs P1 0,02 K0 vs P2 0,00 K0 vs P3 0,00 P1 vs P2 0,03 P1 vs P3 0,00 P2 vs P3 0,03 Discussion This study demonstrates that both alendronate and platelet-rich plasma (PRP) significantly reduce osteoclast numbers following anterior cruciate ligament (ACL) reconstruction using calcaneal tendon grafts in an ovine model. The reduction in osteoclast activity observed in this study supports the central role of bone remodeling modulation in enhancing tendon-to-bone integration, a critical determinant of graft stability and long-term surgical success. Bone healing is a unique regenerative process that recapitulates embryological skeletal development, enabling restoration of pre-injury structure and function 6 . Under physiological conditions, bone integrity is maintained by a dynamic equilibrium between osteoblastic bone formation and osteoclastic bone resorption. Disruption of this balance, particularly under excessive mechanical loading or repetitive cyclic stress, predisposes bone to microdamage and failure 7 . In the context of ACL reconstruction, excessive osteoclastic activity around the bone tunnel may impair osteointegration, leading to tunnel widening and compromised graft fixation. The present findings indicate a statistically significant difference in osteoclast numbers among treatment groups (p = 0.012), confirming that both PRP and alendronate exert a measurable biological effect on bone resorption. These results are consistent with prior work by Sibarani et al. (2024), which demonstrated enhanced osteoblast activity following PRP and alendronate administration, suggesting a coordinated shift toward bone formation 8 . Alendronate, a nitrogen-containing bisphosphonate, inhibits osteoclast-mediated bone resorption through high-affinity binding to hydroxyapatite and subsequent inhibition of farnesyl diphosphate synthase (FPPS), disrupting protein prenylation essential for osteoclast function and survival 9 , 10 . This mechanism selectively suppresses osteoclastic activity while preserving bone mineralization, thereby improving bone mass and strength 11 . Experimental and clinical studies have consistently demonstrated that alendronate reduces fracture risk and increases bone mineral density, with reported fracture risk reductions of up to 50% over three years 12 . In ACL reconstruction models, Lui et al. showed that both local and systemic administration of alendronate significantly reduced bone tunnel resorption, enhanced mineralization, and improved graft–bone integration. Notably, local application minimized systemic effects, whereas systemic administration increased contralateral bone density due to alendronate’s strong skeletal affinity. In the present study, alendronate was administered subcutaneously at a moderate dose (60 µg/kg) 13,14 . Because bilateral extremities were sampled, contralateral effects were not considered confounding, supporting the internal consistency of the findings. PRP functions through a distinct but complementary biological mechanism. Derived from autologous blood, PRP contains a high concentration of growth factors such as TGF-β1, PDGF, IGF-I, FGF, and VEGF, which regulate inflammation, angiogenesis, chemotaxis, and extracellular matrix synthesis 15 , 16 . These mediators are essential for early callus formation and subsequent tissue remodeling. Previous studies have shown that PRP enhances osteogenesis by promoting osteoblast proliferation while reducing osteoclast activity through downregulation of RANKL signaling and activation of β-catenin pathways 17 , 18 . Importantly, evidence suggests a synergistic interaction between alendronate and PRP. Elkaragy and Oman (2013) reported that combined alendronate and PRP treatment produced the highest bone density compared with either intervention alone, indicating that suppression of excessive bone resorption by alendronate may create a favorable microenvironment for PRP-mediated regenerative signaling. This synergy is further supported by the preferential accumulation of alendronate in regions of high bone turnover, such as metaphyseal and trabecular bone surrounding tunnels, where remodeling activity is most pronounced 19 , 20 . The present study also confirmed that sampling location (tibia versus femur) did not significantly influence osteoclast counts (p = 0.211), indicating that the observed effects were independent of anatomical site. This finding strengthens the validity of the histological outcomes and suggests that the intervention effects are broadly applicable within the reconstructed joint environment. From an axiological standpoint, these findings carry both theoretical and clinical relevance. Theoretically, this study reinforces the concept that targeted suppression of osteoclastic activity is a key mechanism underlying successful tendon-to-bone integration. Clinically, the data suggest that alendronate and PRP may serve as promising adjuncts in ACL reconstruction to enhance osteointegration and potentially improve graft stability. Several limitations must be acknowledged. Evaluation was limited to histological and immunohistochemical analyses without molecular validation using techniques such as RT-PCR or Western blotting, which may further elucidate signaling pathways involved. Functional and biomechanical assessments of the reconstructed ACL were not performed, and advanced imaging such as computed tomography was unavailable due to technical constraints. These limitations highlight the need for future studies integrating molecular, biomechanical, and long-term functional outcomes. Conclusion This study demonstrates that both alendronate and platelet-rich plasma (PRP) significantly reduce osteoclast numbers at the tendon–bone interface following anterior cruciate ligament (ACL) reconstruction using a calcaneal tendon graft in a sheep model. Each intervention alone effectively suppressed osteoclastic activity compared with the control group, while the combined administration of alendronate and PRP resulted in the greatest reduction in osteoclast counts, indicating a synergistic effect in modulating bone resorption during the healing process. These findings support the concept that targeted inhibition of excessive osteoclast activity plays a critical role in enhancing tendon-to-bone integration after ACL reconstruction. The combination of alendronate and PRP may create a more favorable biological environment for osteointegration, potentially improving graft stability and surgical outcomes. Further studies incorporating biomechanical, molecular, and long-term functional evaluations are warranted to confirm the translational value of this combined therapeutic approach. Declarations Author Contribution T.S.S. conceived and designed the study and performed the surgical procedures. N.T.W., R.E., and R. were responsible for data collection, including animal handling, postoperative monitoring, tissue harvesting, and histological data acquisition. N.T.W. also wrote the main manuscript text, conducted the histopathological evaluation, and prepared Figures 1–3. R.E. and R. contributed to data interpretation and critically revised the manuscript for important intellectual content. All authors participated in data analysis, reviewed the manuscript critically, and approved the final version of the manuscript for submission. References Morris, J.L., McEwen, P., Letson, H.L., & Dobson, G.P., 2022. Anterior Cruciate Ligament Reconstruction Surgery: Creating a Permissive Healing Phenotype in Military Personnel and Civilians for Faster Recovery. Mil. Med. 187: 1310–1317. doi: 10.1093/milmed/usac093 Zhang, J., Fu, B., Chen, X., Chen, D., & Yang, H., 2020. Protocatechuic acid attenuates anterior cruciate ligament transection–induced osteoarthritis by suppressing osteoclastogenesis. Exp Ther Med 19: 232–240. doi: 10.3892/etm.2019.8189 Liu, X., Zhou, M., Tan, J., Ma, L., Tang, H., He, G., et al., 2024. Inhibition of CX3CL1 by treadmill training prevents osteoclast-induced fibrocartilage complex resorption during TBI healing. Front. Immunol. 14. Shi, S., Duan, H., & Ou, X., 2024. Targeted delivery of anti-osteoporosis therapy: Bisphosphonate-modified nanosystems and composites. Biomed. Pharmacother. 175: 116699. doi: https://doi.org/10.1016/j.biopha.2024.116699 Chalidis, B., Givissis, P., Papadopoulos, P., & Pitsilos, C., 2023. Molecular and Biologic Effects of Platelet-Rich Plasma (PRP) in Ligament and Tendon Healing and Regeneration: A Systematic Review. Int. J. Mol. Sci. 24. doi: 10.3390/ijms24032744 Einhorn, T.A. & Gerstenfeld, L.C. 2015. Fracture healing: Mechanisms and interventions, Nature Reviews Rheumatology . Nature Publishing Group, pp. 45–54. Available at: https://doi.org/10.1038/nrrheum.2014.164 . Fakhry, M. 2013. Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts, World Journal of Stem Cells , 5(4), p.136. Available at: https://doi.org/10.4252/wjsc.v5.i4.136 . Sibarani T, Purwanto B, Mudigdo A, Wasita B. The effect of bisphosphonate and platelet-rich plasma in anterior cruciate ligament reconstruction: an article review. Bali MedJ. 2023. 12(2); 1497–1501 Mary, Aashli & Reddy, S Giridhar & Belliraj, Siva Kumar & Kugabalasooriar, Sanga. (2024). Novel Approaches to Alendronate Delivery Beyond Oral Administration- A Review. Engineered Science . 10.30919/es1274 . Wilkins Parker, L.R., & Preuss, C. V, 2025. Alendronate. Statpearls Publishing , Florida, Treasure Island (FL). Jarusriwanna A, Malisorn S, Tananoo S, Areewong K, Rasamimongkol S, Laoruengthana A. 2024. Efficacy and Safety of Generic Alendronate for Osteoporosis Treatment. Orthop Res Rev. 22;16:85–91. doi: 10.2147/ORR.S445202 . Cosman F, de Beur SJ, LeBoff MS, et al. 2014. Clinician’s guide to prevention and treatment of osteoporosis. Osteoporos Int. 25(10):2359–2381. doi: 10.1007/s00198-014-2794-2 Lui, Pauline Po Yee, Lee, Y.W., Mok, T.Y., & Cheuk, Y.C., 2013. Local administration of alendronate reduced peri-tunnel bone loss and promoted graft-pnnel healing with minimal systemic effect on bone in contralateral knee. J. Orthop. Res. Off. Publ. Orthop. Res. Soc. 31: 1897–1906. doi: 10.1002/jor.22442 Lui, P P Y, Lee, Y.W., Mok, T.Y., Cheuk, Y.C., & Chan, K.M., 2013. Alendronate reduced peri-tunnel bone loss and enhanced tendon graft to bone tunnel healing in anterior cruciate ligament reconstruction. Eur. Cell. Mater. 25: 78–96. doi: 10.22203/ecm.v025a06 Pretorius J, Habash M, Ghobrial B, Alnajjar R, Ellanti P. 2023. Current Status and Advancements in Platelet-Rich Plasma Therapy. Cureus. 15(10):e47176. doi: 10.7759/cureus.47176 . Zhang, Zhixin, Liu, P., Xue, X., Zhang, Zhiyu, Wang, L., Jiang, Y., et al., 2025. The role of platelet-rich plasma in biomedicine: A comprehensive overview. iScience 28: 111705. doi: https://doi.org/10.1016/j.isci.2024.11705 Wang, D., Weng, Y., Guo, S., Zhang, Y., Zhou, T., Zhang, M., et al., 2018. Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling. Int J Mol Med 41: 729–738. doi: 10.3892/ijmm.2017.3258 Mustafa H, Hassan SM, Mohammaed SA, Mohammed MO, Zorab HK, Marif HF. 2024. Influence of platelet-rich plasma on RANKL and IL-1 immunohistochemical expression in periodontitis-related bone cell proliferation and differentiation. The Saudi Dental Journal . 36(12). pp 1593–1600. Elkaragy A and Oman M. 2013. The Efficacy of Using Systemic Alendronate in Combination with Platelet-Rich Plasma in the Osteotomy Implant Site of Osteoporotic Rabbits. Journal of American Science . 9(12). Zebaze, R.M., Libanati, C., Austin, M., Ghasem-Zadeh, A., Hanley, D.A., Zanchetta, J.R., Thomas, T., Boutroy, S., Bogado, C.E., Bilezikian, J.P. & Seeman, E., 2013, ‘Differing effects of denosumab and alendronate on cortical and trabecular bone’, Bone , 57(1), pp. 96–101. https://doi.org/10.1016/j.bone.2013.11.016 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8696555","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584397949,"identity":"227f10ca-36b2-4fc9-8bb8-f81363e50158","order_by":0,"name":"Nugroho Tri Wibowo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACCQY2EMVjf7wBSBlYENbBA9Uiw3DmAEiLBPFabBhuJEAsJQjspZuPPfjYVsfDOPP51Q0/CiQY+Nu7E/DbInMs3XBm22EeZumcsps9QIdJnDm7gYDDcsykebcd4GGTzkm7wQPUYiCRS0hL/jegljoeHskzaTf/EKclhw2ohZlHQoL92G3ibLmRZm44899hHgOeHLbbMgYSPAT9wj4j+dmDD2fq7A3Yjz+7+eaPjRx/ey9+LcgWGoBJYpWDLXxAiupRMApGwSgYQQAAYbtBfKfT/KsAAAAASUVORK5CYII=","orcid":"","institution":"Sebelas Maret University","correspondingAuthor":true,"prefix":"","firstName":"Nugroho","middleName":"Tri","lastName":"Wibowo","suffix":""},{"id":584397951,"identity":"c8163ef0-af41-41e7-a9e9-08260429352a","order_by":1,"name":"Tangkas SMHS Sibarani","email":"","orcid":"","institution":"Soeharso Orthopaedic Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tangkas","middleName":"SMHS","lastName":"Sibarani","suffix":""},{"id":584397952,"identity":"71f99b4f-e6b9-40b0-8e96-e84de40874ac","order_by":2,"name":"Rieva Ermawan","email":"","orcid":"","institution":"Sebelas Maret University","correspondingAuthor":false,"prefix":"","firstName":"Rieva","middleName":"","lastName":"Ermawan","suffix":""},{"id":584397954,"identity":"41dba33f-d96a-42b3-896c-6fe2cea05ac5","order_by":3,"name":"Romaniyanto Romaniyanto","email":"","orcid":"","institution":"Soeharso Orthopaedic Hospital","correspondingAuthor":false,"prefix":"","firstName":"Romaniyanto","middleName":"","lastName":"Romaniyanto","suffix":""}],"badges":[],"createdAt":"2026-01-26 04:53:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8696555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8696555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101852220,"identity":"35df4cae-0ed1-43d5-93b6-d5121f7543ac","added_by":"auto","created_at":"2026-02-04 10:11:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":176542,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin–eosin (H\u0026amp;E) staining shows osteoclast cells (red arrows) at 400× magnification in the K0 (control) group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8696555/v1/e0ff2bbe91931cf11f7d3d2c.png"},{"id":101852128,"identity":"d926ec51-e92e-4102-a914-fc4e37c78012","added_by":"auto","created_at":"2026-02-04 10:11:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":160874,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin–eosin (H\u0026amp;E) staining shows osteoclast cells (red arrows) at 400× magnification in the P1 (Alendronate) grup.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8696555/v1/4e18148697120fe440063f03.png"},{"id":102294911,"identity":"31931d57-c47a-4b59-8d15-c9f4c5df6dff","added_by":"auto","created_at":"2026-02-10 10:04:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":171162,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin–eosin (H\u0026amp;E) staining shows osteoclast cells (red arrows) at 400× magnification in the P2 (PRP) grup.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8696555/v1/6faac544ec0e2faab0535e57.png"},{"id":101852230,"identity":"0307f56b-1212-4621-b96e-304eb9aa9d15","added_by":"auto","created_at":"2026-02-04 10:11:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154826,"visible":true,"origin":"","legend":"\u003cp\u003eHematoxylin–eosin (H\u0026amp;E) staining shows osteoclast cells (red arrows) at 400× magnification in the P3 (PRP + Alendronate) grup.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8696555/v1/90337662f6346dc7a43d6cde.png"},{"id":109544176,"identity":"6f8e4c78-6ec6-4128-9cca-b05a3a296ede","added_by":"auto","created_at":"2026-05-19 10:40:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":899093,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8696555/v1/e0c83b67-bf66-4117-bb1d-a401741301dc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Impact of Graft Choice on Outcomes Following Pediatric Medial Patellofemoral Ligament Reconstruction: A Systematic Review","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBone tissue healing and tendon-to-bone integration are complex processes influenced by multiple biological factors. Limited blood supply to ligaments, such as in anterior cruciate ligament (ACL) injuries, impairs spontaneous healing. If left untreated, such injuries can lead to knee instability and secondary cartilage damage, including meniscal injury. A key regulator of bone remodeling is the osteoclast, a multinucleated cell responsible for bone resorption. Excessive osteoclast activity at the tendon insertion site can disrupt tendon\u0026ndash;bone integration, weaken the healing interface, and ultimately compromise the success of ligament reconstruction \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eModulating osteoclast activity is a promising approach to improve tendon-to-bone healing. Studies have shown that suppressing osteoclast function enhances bone formation at the repair site. Alendronate, a bisphosphonate, binds to hydroxyapatite crystals and induces osteoclast apoptosis, thereby reducing bone resorption and promoting faster bone regeneration\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In addition, platelet-rich plasma (PRP), a biologic product rich in growth factors such as PDGF and IGF-1, has been reported to suppress osteoclast activity while stimulating osteoblast-mediated bone formation\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough several studies have investigated the effects of alendronate and PRP individually on osteoclast activity, research on their combined use remains limited. This study aims to evaluate the effects of alendronate and PRP on osteoclast levels in a sheep ACL rupture model, providing new insights into biologically driven strategies to accelerate and optimize ACL injury healing and tendon-to-bone integration.\u003c/p\u003e"},{"header":"Material and Method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStudy Design\u003c/h2\u003e\n\u003cp\u003eThis experimental study employed a post-test only control group design using sheep (Ovis aries) as the animal model. Twenty-eight animals were randomly allocated into four groups (n\u0026thinsp;=\u0026thinsp;7 per group):\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eControl Group (K0): ACL reconstruction using calcaneal tendon graft, followed by subcutaneous administration of 0.9% NaCl.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eTreatment Group 1 (P1): ACL reconstruction plus weekly subcutaneous injections of alendronate (60 \u0026micro;g/kg body weight) for six weeks.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eTreatment Group 2 (P2): ACL reconstruction plus local administration of platelet-rich plasma (PRP).\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eTreatment Group 3 (P3): ACL reconstruction plus combined PRP application and weekly alendronate injections (60 \u0026micro;g/kg) for six weeks.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAll procedures were conducted in accordance with institutional animal welfare regulations and international research ethics standards.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eStudy Setting and Duration\u003c/h3\u003e\n\u003cp\u003eThe study was conducted at the Animal Laboratory, Faculty of Veterinary Medicine, Universitas Gadjah Mada (Yogyakarta, Indonesia). Histopathological analysis was performed at the Department of Anatomical Pathology, Faculty of Medicine, Universitas Sebelas Maret (Surakarta, Indonesia). The experiment was conducted from April 2025 until completion.\u003c/p\u003e\n\u003ch3\u003eExperimental Animals\u003c/h3\u003e\n\u003cp\u003eHealthy, skeletally mature male sheep (Ovis aries), aged 2 years and weighing approximately 20 kg, were included. Animals with degenerative joint disease or systemic illness were excluded. Any animal that died during the study period was recorded and excluded from analysis.\u003c/p\u003e\n\u003ch3\u003eSample Size Calculation\u003c/h3\u003e\n\u003cp\u003eThe minimum sample size was calculated using the Notoadmodjo formula (2018):\u003c/p\u003e\n\u003cp\u003e(𝑡\u0026minus;1) (𝑛\u0026minus;1) \u0026gt; 15\u003c/p\u003e\n\u003cp\u003ewhere t\u0026thinsp;=\u0026thinsp;number of groups and n\u0026thinsp;=\u0026thinsp;sample size per group. With four groups, a minimum of six animals per group was required. To account for a potential 10% dropout, the sample size per group was increased to seven, yielding a total of 28 animals.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003ePreoperative Preparation\u003c/h2\u003e\n\u003cp\u003eAll sheep underwent a 7-day acclimatization period under standard housing conditions and were assessed for general health status. Animals were then randomly assigned to one of the four groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSurgical Procedure\u003c/h3\u003e\n\u003cp\u003eAnterior cruciate ligament (ACL) reconstruction was performed under general anesthesia and sterile conditions. Following a medial parapatellar approach, the native ACL was transected. Autologous calcaneal tendon was harvested, and femoral and tibial tunnels were created anatomically to match the graft diameter. The graft was positioned at the ACL footprint, tensioned at 30\u0026deg; knee flexion, and fixed with a 6-mm biodegradable interference screw.\u003c/p\u003e\n\u003ch3\u003ePRP Preparation and Application\u003c/h3\u003e\n\u003cp\u003eAutologous whole blood was collected and anticoagulated with sodium citrate. PRP was prepared using a two-step centrifugation protocol to achieve a platelet concentration of at least four times baseline. The prepared PRP was applied intraoperatively around the tendon-bone interface after graft fixation.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eAlendronate Administration\u003c/h2\u003e\n\u003cp\u003eAlendronate was administered subcutaneously at a dose of 60 \u0026micro;g/kg once weekly for six consecutive weeks, starting on the day of surgery.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003ePostoperative Care and Follow-Up\u003c/h2\u003e\n\u003cp\u003eAnimals were housed under identical environmental and feeding conditions. They were observed daily for wound healing, mobility, and signs of infection or distress.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eTissue Harvesting and Histological Analysis\u003c/h2\u003e\n\u003cp\u003eAt 12 weeks post-surgery, animals were euthanized humanely. The femur\u0026ndash;tibia complex was harvested, and tissue samples from the bone\u0026ndash;tendon junction were fixed in 10% formalin, decalcified, embedded in paraffin, sectioned, and stained with hematoxylin\u0026ndash;eosin (H\u0026amp;E). Osteoclasts were identified as multinucleated cells along the bone surface and counted in five randomly selected high-power fields (HPFs) per section by two independent, blinded pathologists.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eOutcome Measures\u003c/h2\u003e\n\u003cp\u003eThe primary outcome measure was the number of osteoclasts at the tendon\u0026ndash;bone interface.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n\u003cp\u003eData distribution was assessed using the Shapiro\u0026ndash;Wilk test, and homogeneity of variance was evaluated with Levene\u0026rsquo;s test. If data were normally distributed and homogeneous, one-way ANOVA was applied followed by Tukey\u0026rsquo;s post hoc test. Non-normally distributed data were analyzed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparison test. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analyses were performed using RStudio (version 2024.12.1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eEthical Considerations\u003c/h2\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Faculty of Medicine, Universitas Sebelas Maret (Approval No. [insert number]). All procedures adhered to the ARRIVE guidelines and the National Research Council\u0026rsquo;s Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eHistological examination was successfully performed on all 28 specimens following a 12-week observation period after anterior cruciate ligament reconstruction. Osteoclast evaluation was conducted on sections obtained from the bone\u0026ndash;tendon junction and stained using hematoxylin and eosin (H\u0026amp;E). Quantitative assessment of osteoclasts was then performed and the results were compared among the four experimental groups.\u003c/p\u003e \u003cp\u003eThe mean (\u0026plusmn;\u0026thinsp;standard deviation) osteoclast counts were highest in the control group (K0: 12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52). A marked reduction in osteoclast numbers was observed in all intervention groups, with sequentially lower values in the alendronate group (P1: 5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09), the PRP group (P2: 3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61), and the lowest count in the combined alendronate and PRP group (P3: 1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99). These histological differences across groups are illustrated in Figs.\u0026nbsp;1\u0026ndash;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eAssessment of data distribution using the Shapiro\u0026ndash;Wilk test revealed that osteoclast count data in all groups were not normally distributed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, Levene\u0026rsquo;s test demonstrated heterogeneity of variances among groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, the data did not meet the assumptions required for parametric analysis, and nonparametric statistical methods were applied. Group comparisons were performed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s post hoc test for pairwise comparisons.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean of osteoclast number after intervention\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean \u0026plusmn; Standard Deviation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNormality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHomogeneity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.00\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0,0134\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.43\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.71\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.71\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Kruskal\u0026ndash;Wallis analysis showed a statistically significant difference in osteoclast counts among the four groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Further post hoc analysis using Dunn\u0026rsquo;s test demonstrated significant differences between all pairwise group comparisons (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as summarized in Table\u0026nbsp;2. These findings indicate that both single-agent interventions (alendronate or PRP) and the combined therapy significantly reduced osteoclast numbers at the tendon\u0026ndash;bone interface compared with the control group.\u003c/p\u003e \u003cp\u003eOverall, the histological results suggest that modulation of bone remodeling through alendronate and PRP effectively suppresses osteoclastic activity following ACL reconstruction. The most pronounced reduction in osteoclast counts was consistently observed in the combined treatment group (P3), supporting a potential synergistic effect of alendronate and PRP in promoting a more favorable biological environment for tendon-to-bone healing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e. \u003cb\u003eTable\u0026nbsp;2\u003c/b\u003e Statistical analysis results of \u003cem\u003epost hoc\u003c/em\u003e Dunn\u0026rsquo;s test on number of osteoclast\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK0 vs P1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK0 vs P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK0 vs P3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1 vs P2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1 vs P3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2 vs P3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0,03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that both alendronate and platelet-rich plasma (PRP) significantly reduce osteoclast numbers following anterior cruciate ligament (ACL) reconstruction using calcaneal tendon grafts in an ovine model. The reduction in osteoclast activity observed in this study supports the central role of bone remodeling modulation in enhancing tendon-to-bone integration, a critical determinant of graft stability and long-term surgical success.\u003c/p\u003e \u003cp\u003eBone healing is a unique regenerative process that recapitulates embryological skeletal development, enabling restoration of pre-injury structure and function\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Under physiological conditions, bone integrity is maintained by a dynamic equilibrium between osteoblastic bone formation and osteoclastic bone resorption. Disruption of this balance, particularly under excessive mechanical loading or repetitive cyclic stress, predisposes bone to microdamage and failure\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In the context of ACL reconstruction, excessive osteoclastic activity around the bone tunnel may impair osteointegration, leading to tunnel widening and compromised graft fixation.\u003c/p\u003e \u003cp\u003eThe present findings indicate a statistically significant difference in osteoclast numbers among treatment groups (p\u0026thinsp;=\u0026thinsp;0.012), confirming that both PRP and alendronate exert a measurable biological effect on bone resorption. These results are consistent with prior work by Sibarani et al. (2024), which demonstrated enhanced osteoblast activity following PRP and alendronate administration, suggesting a coordinated shift toward bone formation\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlendronate, a nitrogen-containing bisphosphonate, inhibits osteoclast-mediated bone resorption through high-affinity binding to hydroxyapatite and subsequent inhibition of farnesyl diphosphate synthase (FPPS), disrupting protein prenylation essential for osteoclast function and survival\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This mechanism selectively suppresses osteoclastic activity while preserving bone mineralization, thereby improving bone mass and strength\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Experimental and clinical studies have consistently demonstrated that alendronate reduces fracture risk and increases bone mineral density, with reported fracture risk reductions of up to 50% over three years\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn ACL reconstruction models, Lui et al. showed that both local and systemic administration of alendronate significantly reduced bone tunnel resorption, enhanced mineralization, and improved graft\u0026ndash;bone integration. Notably, local application minimized systemic effects, whereas systemic administration increased contralateral bone density due to alendronate\u0026rsquo;s strong skeletal affinity. In the present study, alendronate was administered subcutaneously at a moderate dose (60 \u0026micro;g/kg)\u003csup\u003e13,14\u003c/sup\u003e. Because bilateral extremities were sampled, contralateral effects were not considered confounding, supporting the internal consistency of the findings.\u003c/p\u003e \u003cp\u003ePRP functions through a distinct but complementary biological mechanism. Derived from autologous blood, PRP contains a high concentration of growth factors such as TGF-β1, PDGF, IGF-I, FGF, and VEGF, which regulate inflammation, angiogenesis, chemotaxis, and extracellular matrix synthesis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. These mediators are essential for early callus formation and subsequent tissue remodeling. Previous studies have shown that PRP enhances osteogenesis by promoting osteoblast proliferation while reducing osteoclast activity through downregulation of RANKL signaling and activation of β-catenin pathways\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImportantly, evidence suggests a synergistic interaction between alendronate and PRP. Elkaragy and Oman (2013) reported that combined alendronate and PRP treatment produced the highest bone density compared with either intervention alone, indicating that suppression of excessive bone resorption by alendronate may create a favorable microenvironment for PRP-mediated regenerative signaling. This synergy is further supported by the preferential accumulation of alendronate in regions of high bone turnover, such as metaphyseal and trabecular bone surrounding tunnels, where remodeling activity is most pronounced\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe present study also confirmed that sampling location (tibia versus femur) did not significantly influence osteoclast counts (p\u0026thinsp;=\u0026thinsp;0.211), indicating that the observed effects were independent of anatomical site. This finding strengthens the validity of the histological outcomes and suggests that the intervention effects are broadly applicable within the reconstructed joint environment.\u003c/p\u003e \u003cp\u003eFrom an axiological standpoint, these findings carry both theoretical and clinical relevance. Theoretically, this study reinforces the concept that targeted suppression of osteoclastic activity is a key mechanism underlying successful tendon-to-bone integration. Clinically, the data suggest that alendronate and PRP may serve as promising adjuncts in ACL reconstruction to enhance osteointegration and potentially improve graft stability.\u003c/p\u003e \u003cp\u003eSeveral limitations must be acknowledged. Evaluation was limited to histological and immunohistochemical analyses without molecular validation using techniques such as RT-PCR or Western blotting, which may further elucidate signaling pathways involved. Functional and biomechanical assessments of the reconstructed ACL were not performed, and advanced imaging such as computed tomography was unavailable due to technical constraints. These limitations highlight the need for future studies integrating molecular, biomechanical, and long-term functional outcomes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that both alendronate and platelet-rich plasma (PRP) significantly reduce osteoclast numbers at the tendon\u0026ndash;bone interface following anterior cruciate ligament (ACL) reconstruction using a calcaneal tendon graft in a sheep model. Each intervention alone effectively suppressed osteoclastic activity compared with the control group, while the combined administration of alendronate and PRP resulted in the greatest reduction in osteoclast counts, indicating a synergistic effect in modulating bone resorption during the healing process.\u003c/p\u003e \u003cp\u003eThese findings support the concept that targeted inhibition of excessive osteoclast activity plays a critical role in enhancing tendon-to-bone integration after ACL reconstruction. The combination of alendronate and PRP may create a more favorable biological environment for osteointegration, potentially improving graft stability and surgical outcomes. Further studies incorporating biomechanical, molecular, and long-term functional evaluations are warranted to confirm the translational value of this combined therapeutic approach.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eT.S.S. conceived and designed the study and performed the surgical procedures. N.T.W., R.E., and R. were responsible for data collection, including animal handling, postoperative monitoring, tissue harvesting, and histological data acquisition. N.T.W. also wrote the main manuscript text, conducted the histopathological evaluation, and prepared Figures 1\u0026ndash;3. R.E. and R. contributed to data interpretation and critically revised the manuscript for important intellectual content. All authors participated in data analysis, reviewed the manuscript critically, and approved the final version of the manuscript for submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMorris, J.L., McEwen, P., Letson, H.L., \u0026amp; Dobson, G.P., 2022. Anterior Cruciate Ligament Reconstruction Surgery: Creating a Permissive Healing Phenotype in Military Personnel and Civilians for Faster Recovery. \u003cem\u003eMil. Med.\u003c/em\u003e 187: 1310\u0026ndash;1317. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/milmed/usac093\u003c/span\u003e\u003cspan address=\"10.1093/milmed/usac093\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J., Fu, B., Chen, X., Chen, D., \u0026amp; Yang, H., 2020. Protocatechuic acid attenuates anterior cruciate ligament transection\u0026ndash;induced osteoarthritis by suppressing osteoclastogenesis. \u003cem\u003eExp Ther Med\u003c/em\u003e 19: 232\u0026ndash;240. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/etm.2019.8189\u003c/span\u003e\u003cspan address=\"10.3892/etm.2019.8189\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, X., Zhou, M., Tan, J., Ma, L., Tang, H., He, G., et al., 2024. Inhibition of CX3CL1 by treadmill training prevents osteoclast-induced fibrocartilage complex resorption during TBI healing. \u003cem\u003eFront. Immunol.\u003c/em\u003e 14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, S., Duan, H., \u0026amp; Ou, X., 2024. Targeted delivery of anti-osteoporosis therapy: Bisphosphonate-modified nanosystems and composites. \u003cem\u003eBiomed. Pharmacother.\u003c/em\u003e 175: 116699. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopha.2024.116699\u003c/span\u003e\u003cspan address=\"10.1016/j.biopha.2024.116699\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChalidis, B., Givissis, P., Papadopoulos, P., \u0026amp; Pitsilos, C., 2023. Molecular and Biologic Effects of Platelet-Rich Plasma (PRP) in Ligament and Tendon Healing and Regeneration: A Systematic Review. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e 24. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ijms24032744\u003c/span\u003e\u003cspan address=\"10.3390/ijms24032744\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEinhorn, T.A. \u0026amp; Gerstenfeld, L.C. 2015. Fracture healing: Mechanisms and interventions, \u003cem\u003eNature Reviews Rheumatology\u003c/em\u003e. Nature Publishing Group, pp. 45\u0026ndash;54. Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrrheum.2014.164\u003c/span\u003e\u003cspan address=\"10.1038/nrrheum.2014.164\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFakhry, M. 2013. Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts, \u003cem\u003eWorld Journal of Stem Cells\u003c/em\u003e, 5(4), p.136. Available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4252/wjsc.v5.i4.136\u003c/span\u003e\u003cspan address=\"10.4252/wjsc.v5.i4.136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSibarani T, Purwanto B, Mudigdo A, Wasita B. The effect of bisphosphonate and platelet-rich plasma in anterior cruciate ligament reconstruction: an article review. Bali MedJ. 2023. 12(2); 1497\u0026ndash;1501\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMary, Aashli \u0026amp; Reddy, S Giridhar \u0026amp; Belliraj, Siva Kumar \u0026amp; Kugabalasooriar, Sanga. (2024). Novel Approaches to Alendronate Delivery Beyond Oral Administration- A Review. \u003cem\u003eEngineered Science\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.30919/es1274\u003c/span\u003e\u003cspan address=\"10.30919/es1274\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkins Parker, L.R., \u0026amp; Preuss, C. V, 2025. Alendronate. \u003cem\u003eStatpearls Publishing\u003c/em\u003e, Florida, Treasure Island (FL).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJarusriwanna A, Malisorn S, Tananoo S, Areewong K, Rasamimongkol S, Laoruengthana A. 2024. Efficacy and Safety of Generic Alendronate for Osteoporosis Treatment. \u003cem\u003eOrthop Res Rev.\u003c/em\u003e 22;16:85\u0026ndash;91. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2147/ORR.S445202\u003c/span\u003e\u003cspan address=\"10.2147/ORR.S445202\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCosman F, de Beur SJ, LeBoff MS, et al. 2014. Clinician\u0026rsquo;s guide to prevention and treatment of osteoporosis. \u003cem\u003eOsteoporos Int.\u003c/em\u003e 25(10):2359\u0026ndash;2381. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00198-014-2794-2\u003c/span\u003e\u003cspan address=\"10.1007/s00198-014-2794-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLui, Pauline Po Yee, Lee, Y.W., Mok, T.Y., \u0026amp; Cheuk, Y.C., 2013. Local administration of alendronate reduced peri-tunnel bone loss and promoted graft-pnnel healing with minimal systemic effect on bone in contralateral knee. \u003cem\u003eJ. Orthop. Res. Off. Publ. Orthop. Res. Soc.\u003c/em\u003e 31: 1897\u0026ndash;1906. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/jor.22442\u003c/span\u003e\u003cspan address=\"10.1002/jor.22442\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLui, P P Y, Lee, Y.W., Mok, T.Y., Cheuk, Y.C., \u0026amp; Chan, K.M., 2013. Alendronate reduced peri-tunnel bone loss and enhanced tendon graft to bone tunnel healing in anterior cruciate ligament reconstruction. \u003cem\u003eEur. Cell. Mater.\u003c/em\u003e 25: 78\u0026ndash;96. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22203/ecm.v025a06\u003c/span\u003e\u003cspan address=\"10.22203/ecm.v025a06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePretorius J, Habash M, Ghobrial B, Alnajjar R, Ellanti P. 2023. Current Status and Advancements in Platelet-Rich Plasma Therapy. \u003cem\u003eCureus.\u003c/em\u003e 15(10):e47176. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7759/cureus.47176\u003c/span\u003e\u003cspan address=\"10.7759/cureus.47176\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Zhixin, Liu, P., Xue, X., Zhang, Zhiyu, Wang, L., Jiang, Y., et al., 2025. The role of platelet-rich plasma in biomedicine: A comprehensive overview. \u003cem\u003eiScience\u003c/em\u003e 28: 111705. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.isci.2024.11705\u003c/span\u003e\u003cspan address=\"10.1016/j.isci.2024.11705\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, D., Weng, Y., Guo, S., Zhang, Y., Zhou, T., Zhang, M., et al., 2018. Platelet-rich plasma inhibits RANKL-induced osteoclast differentiation through activation of Wnt pathway during bone remodeling. \u003cem\u003eInt J Mol Med\u003c/em\u003e 41: 729\u0026ndash;738. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/ijmm.2017.3258\u003c/span\u003e\u003cspan address=\"10.3892/ijmm.2017.3258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMustafa H, Hassan SM, Mohammaed SA, Mohammed MO, Zorab HK, Marif HF. 2024. Influence of platelet-rich plasma on RANKL and IL-1 immunohistochemical expression in periodontitis-related bone cell proliferation and differentiation. \u003cem\u003eThe Saudi Dental Journal\u003c/em\u003e. 36(12). pp 1593\u0026ndash;1600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElkaragy A and Oman M. 2013. The Efficacy of Using Systemic Alendronate in Combination with Platelet-Rich Plasma in the Osteotomy Implant Site of Osteoporotic Rabbits. \u003cem\u003eJournal of American Science\u003c/em\u003e. 9(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZebaze, R.M., Libanati, C., Austin, M., Ghasem-Zadeh, A., Hanley, D.A., Zanchetta, J.R., Thomas, T., Boutroy, S., Bogado, C.E., Bilezikian, J.P. \u0026amp; Seeman, E., 2013, \u0026lsquo;Differing effects of denosumab and alendronate on cortical and trabecular bone\u0026rsquo;, \u003cem\u003eBone\u003c/em\u003e, 57(1), pp. 96\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bone.2013.11.016\u003c/span\u003e\u003cspan address=\"10.1016/j.bone.2013.11.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Anterior cruciate ligament reconstruction, alendronate, platelet-rich plasma, osteoclast, tendon-to-bone healing, sheep model","lastPublishedDoi":"10.21203/rs.3.rs-8696555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8696555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSuccessful anterior cruciate ligament (ACL) reconstruction is highly dependent on effective tendon-to-bone healing. Excessive osteoclast activity at the bone\u0026ndash;tendon interface can disrupt bone remodeling, impair graft integration, and increase the risk of graft failure. Modulation of osteoclast activity using pharmacological and biological agents may enhance osteointegration after ACL reconstruction.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThis study aimed to evaluate the effects of alendronate and platelet-rich plasma (PRP), administered alone and in combination, on osteoclast levels at the bone\u0026ndash;tendon junction following ACL reconstruction using a calcaneal tendon graft in a sheep model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA randomized controlled experimental study was conducted on 28 sheep subjected to ACL A randomized controlled experimental study was conducted on 28 skeletally mature sheep undergoing ACL reconstruction with a calcaneal tendon autograft. Animals were randomly assigned to four groups (n\u0026thinsp;=\u0026thinsp;7 per group): control (K0), alendronate-treated (P1), PRP-treated (P2), and combined alendronate plus PRP-treated (P3). Alendronate was administered subcutaneously at a dose of 60 \u0026micro;g/kg weekly for six weeks, while PRP was applied locally at the tendon\u0026ndash;bone interface intraoperatively. Histological evaluation was performed 12 weeks postoperatively using hematoxylin\u0026ndash;eosin staining. Osteoclasts were identified and quantified at the tendon\u0026ndash;bone junction. Data were analyzed using the Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s post hoc test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ehere was a significant difference in osteoclast counts among groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The control group exhibited the highest mean osteoclast count (12.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52), followed by the alendronate group (5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09), PRP group (3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61), and the lowest count in the combined treatment group (1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99). Post hoc analysis demonstrated significant differences between all group comparisons (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the most pronounced reduction observed in the alendronate plus PRP group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAlendronate and PRP significantly reduce osteoclast activity at the tendon\u0026ndash;bone interface following ACL reconstruction, with a synergistic effect observed when both therapies are combined. These findings suggest that combined alendronate and PRP therapy may enhance tendon-to-bone healing and improve biological conditions for graft integration after ACL reconstruction.\u003c/p\u003e","manuscriptTitle":"The Impact of Graft Choice on Outcomes Following Pediatric Medial Patellofemoral Ligament Reconstruction: A Systematic Review","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 10:09:07","doi":"10.21203/rs.3.rs-8696555/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"75f255cf-f38f-48d1-9483-617e7fc24851","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-19T10:28:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T06:11:10+00:00","index":63,"fulltext":""},{"type":"reviewerAgreed","content":"14817305166227270670606662725769959842","date":"2026-05-12T13:14:13+00:00","index":62,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T10:40:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 10:09:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8696555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8696555","identity":"rs-8696555","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}